Composition Exhibiting a von Willebrand Factor (vWF) Protease Activity Comprising a Polypeptide Chain with the Amino Acid Sequence AAGGILHLELLV

ABSTRACT

The invention relates to vWF cleaving entities having a molecular weight of 180 kD, 170 kD, 160 kD, 120 kD or 110 kD and an N-terminal amino acid sequence of AAGGILHLELLV, vWF cleaving complexes and methods for their production.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/247,116 filed Apr. 7, 2014, which is a divisional of U.S. applicationSer. No. 12/032,553 filed Feb. 15, 2008 (now U.S. Pat. No. 8,703,426issued Apr. 22, 2014), which is a divisional of U.S. application Ser.No. 11/166,288 filed Jun. 23, 2005 (now U.S. Pat. No. 7,501,117 issuedMar. 10, 2009), which is a continuation of U.S. application Ser. No.09/833,328 filed Apr. 12, 2001 (now U.S. Pat. No. 6,926,894 issued Aug.9, 2005), which is a continuation-in-part of application Ser. No.09/721,254 filed Nov. 22, 2000 (abandoned), all of which are hereinincorporated by reference.

FIELD OF THE INVENTION

The invention relates to a vWF protease-containing composition whichincludes a potypeptide comprising the amino add sequence AAGGILHLELLV,as well as to nucleotide sequences coding for such a polypeptide. Itfurther relates to methods for increasing the stability of the vWFprotease.

BACKGROUND OF THE INVENTION

vWF is a glycoprotein circulating in plasma as a series of multimersranging in size from about 500 to 20,000 kD. Multimeric forms of vWF arecomposed of 250 kD polypeptide subunits linked together by disulfidebonds. vWF mediates the initial platelet adhesion to the subendotheliumof a damaged vessel wall, though only the largest multimers appear toexhibit haemostatic activity. Such vWF multimers having large molecularmasses are stored in the Weibel Palade bodies of endothelial cells, andit is believed that endothelial cells secrete these large polymericforms of vWF. Those forms of vWF which have a low molecular weight (lowmolecular weight or LMW vWF) are believed to arise from proteolyticcleavage of the larger multimers.

A small portion of the vWF present in normal plasma circulates as 189,176 and 140 kD fragments resulting from proteolytic degradation of vWFin vivo, the 140 kD fragment being derived from the N-terminal region,and the 176 kD fragment from the C-terminal region of the subunit. WhenLMW forms of vWF are isolated from normal human plasma and subjected toSDS-PAGE (polyacrylamide gel electrophoreses) after disulfide reduction,an unusually high portion of vWF fragments are found. This finding iscompatible with the view that LMW forms of vWF have been partially orpredominantly derived from large multimers by proteolytic degradation.

The proteolytic degradation of vWF is a physiological process in healthyindividuals, yet in patients suffering from von Willebrand disease (vWD)type 2A it may be accelerated, and as a consequence these patients lackthe vWF multimers with the largest molecular masses. A lack of large vWFmultimers and an increased level of proteolytic fragments are alsoobserved in acquired von Willebrand disease (vWD) associated withmyeloproliferation syndrome, indicating increased in vivo proteolysis inthis condition as well.

In patients with thrombotic thrombocytopenic purpura (TTP), on the otherhand, unusually large vWF multimers are detected, and increased vWFbinding to platelets has been demonstrated in these patients (Wake et.al., New Engl. J. Med., 1982, 307, pp. 1432-1435). Familial TIP isassociated with a severe congenital deficiency of vWF protease, whilethe presence of vWF-cleaving proteases inhibiting autoantibodies hasbeen observed in patients with non-familial TTP.

The large multimers of vWF associated with TTP normally disappear aftera patient is transfused with normal fresh frozen plasma. Presently,plasma exchange is the most important treatment for TTP, althoughsignificant side effects have been reported with this therapy. Theexistence of a severe congenital deficiency of vWF protease has beenestablished in patients with familial TTP and the presence of avWF-cleaving protease inhibiting autoantibodies has been observed inpatients with non-familial TTP.

Several proteases have been shown to be able to cleave vWF, therebyimpairing its binding affinity for platelets. However, in vitro thecleavage of vWF with these proteases in each case results in cleavageproducts different from the fragments derived from in vivo cleavage.

Thus, for example, while plasmin is capable of cleaving several peptidebonds in vWF, plasmin-treated vWF retains a high molecular weight coreregion retaining about 70% of its platelet agglutinating activity(determined as ristocetin cofactor). A 34 kD peptide is split from theN-termini of individual vWF subunits in the early stages of plasmintreatment, and epitope mapping of such plasmin-induced fragments showthat these fragments originated from regions of the vWF subunit that aredifferent from the vWF fragments present in circulating plasma.

Porcine pancreatic elastase and various serine proteases released fromhuman leukocytes have also been shown to degrade vWF proteolyticallywith a resultant loss of large multimers. Epitope mapping of thedegradation products again indicates that these fragments also differfrom those present in normal plasma and in vWD type 2A. In addition, acalpain-like protease released from human platelets has been shown todegrade large vWF multimers and to create vWF fragments similar to thoseobserved in vivo.

SUMMARY OF THE INVENTION

We have isolated a composition exhibiting vWF protease activity that iscapable of proteolytically processing vWF in a physiological manner.Said composition comprises at least one single peptide chain having amolecular weight between 190 kD and 100 kD as determined by SDS PAGE andcomprises the sequence AAGGILHLELLV. This amino acid sequence is locatedat the N-terminus of the peptide chain. The composition comprising thesequence AAGGHILHLELLV can also be used for isolation, detection orpurification of proteins, i.e. von Willebrand Factor.

Furthermore, an isolated polypeptide having a molecular weight between190 kD and 100 kD according to SDS-PAGE and comprising the sequenceAAGGILHLELLV is also provided. This sequence is preferably directlyfollowed by the sequence AVG, which is preferably followed by thesequence PDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTI.

Another aspect of the present invention is a method of purifying vonWillebrand factor comprising contacting a solution containing vonWillebrand factor with a substrate comprising the amino acid sequenceAAGGILHLELLV under conditions sufficient to bind von Willebrand factorto the substrate.

The present invention further comprises, a method of treating thromboticdiseases using a polypeptide of the present invention, as shown in FIG.2.

In addition, the present invention includes a method of processingrecombinantly produced vWF through the use of the vWF protease of thepresent invention, in order to produce a vWF product.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the schematic purification scheme of the vWF proteasecontaining composition of the present invention.

FIG. 2 shows the partial nucleotide and amino acid sequence of the vWFprotease of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

A composition is provided containing a vWF protease consisting of apolypeptide chain with an apparent molecular weight in SDS-PAGE ofaround 180 kD, around 170 kD, around 160 kD, around 120 kD, around 110kD or mixtures of these chains, said chain comprising an amino acidsequence AAGGILHLELLV. Alternatively, this amino acid sequence can bedirectly followed by the amino acid sequence AVG. Furthermore, thissequence is followed by the sequencePDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTI.

The SOS-PAGE was performed under reducing conditions. As is well knownin the art, molecular weight determination using SDS PAGE results in thedetection of apparent molecular masses, which may be different from themolecular masses of the native, non-denatured protein.

Based on a computer search of sequence homologies, the vWF proteaseaccording to the invention shows high homology to the group ofdisintegrin and metalloproteinases (ADAM). Members of this group shareseveral distinct protein modules, including a protease domain, adisintegrin domain, a cysteine-rich region and an EGF repeat (Tang B Land Hong W., FEBS, 1999, 445, pp. 223-225). The nucleotide and aminoacid sequence as shown in FIG. 2 comprises at least 4 axons of theprotease portion of the vWF protease of the present invention.

Analysis of non-denatured material by mass spectrometry showed verybroad peaks of high molecular weight. No prominent bands wereidentified. This finding is in agreement with appearance, in gelfiltration experiments, of 80 kD bands close to the void volume of.Sephacryl® S-300, suggesting that the proteins in this preparation tendto polymerize under physiologic conditions (a property of clusterin).

The AAGGILHLELLV sequence is located at the N-terminal region of theprotein. Shortening of the peptide chain occurs at the C-terminus or viaendoproteolytical cleavages. The composition according to the presentinvention contains a vWF cleaving protease that is expressed as a singlechain protein.

Preferably, the composition according to the present invention furthercomprises Ca²⁺, Sr²⁺ and/or Ba²⁺ ions. The preparation may comprisecalcium ions in a. concentration of about 1 to 10⁶ ions per polypeptidemolecule with vWF protease activity. The preparation according to thepresent invention contains vWF protease activity in an essentiallypurified form. Preferably, the purified protease preparation from plasmacontains between 0.001% and 1%, preferably 0.002% of the initial amountof plasma protein, and between 1% and 5%, preferably 2.3% of the initialenzyme activity, which has been partially inactivated during thepurification procedure. Preferably, the purity is as high as therelative proportions of polypeptide chains with the vWF proteaseactivity present in an amount of above 50%, especially above 80%, mostpreferred about 90%, of total protein compared to the vWF proteaseactivity in plasma. Preferably, the preparation according to the presentinvention is essentially free of vWF or vWF fragments, i.e. having a vWFcontent of below 5%, preferably below the detection limit of an assay todetect vWF.

The peptides containing the amino acid sequence AAGGILHLELLV orAAGGILHLELLVAVG or AAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTI or RRAAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTI can also be used as tool for detecting proteins bindingto the vWF protease or target sites for ligand development fordetecting, isolating and purifying proteins that bind to the vWFprotease. Preferably the protein to be detected or purified is vWF.

These ligands can for example be peptides or peptidomimetics capable tobind proteins binding to the vWF protease or binding domainsincorporated into antibodies or antibody derivatives (for example singlechain antibodies, miniantibodies, bispecific antibodies, diabodiesetc.).

Furthermore, the peptide having the amino acid sequence AAGGILHLELLV orAAGGILHLELLVAVG or AAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTI or RRAAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTI can also be used for the development of anit-vWFprotease antibodies using techniques as known from the art. Thedevelopment of these antibodies or antibody derivatives orpeptidomimetics can be accomplished according methods known to the priorart (Greer J. et al., J. Med. Chem., 1994, Vol. 37, pp. 1035-1054;Harlow E. and Lane D., in “Antibodies. A Laboratory manual”, Cold SpringHarbor Laboratory, 1988, Esser C. and Radbruchj A. Annu. Rev. Immunol.,1990, vol. 8, pp. 717-735; Kemp D. S., 1990, Trends Biotechnol., pp.249-255).

The present invention relates also to single polypeptide chains havingan apparent molecular weight in reduced SDS-PAGE of between 190 kD and100 kD, preferably about 180 kD, more preferably about 170 kD, in aparticularly preferred embodiment about 160 kD, preferably 120 kD andmost preferably about 110 kD, comprising an N-terminal amino acidsequence AAGGILHLELLV. This sequence is preferably directly followed bythe sequence AVG, which is then preferably followed by the sequencePDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTI. In contrastto the protease entities described by Furlan et al. (1996) and Tsai(Blood 87 (10) (1996), pp. 4235-4244) the vWF multimerase entitiesaccording to the present invention are much smaller than the entitiesdescribed in these documents (around 300 kD (Furlan et al.), and 200 kD(Tsai), respectively).

The proteolytic entities provided with the present invention comprise aphysiological vWF cleaving activity which is defined by (1) the cleavingvWF at the peptide bond 842Ty r-843Met, (2) having a direct proteolyticactivity which converts vWF having a singlet structure to vWF having asatellite structure, and (3) retaining activity in the presence of aserine protease inhibitor such as diisopropyl fluorophosphate (DFP) andin the presence of a calpain protease inhibitor such as carbobenzyloxy(Z) peptidyl diazomethylketone inhibitor (Z-Leu-Leu-Tyr-CHN₂). Theproteolytic entities provided with the present invention may also actindirectly via another effector protein, for example a protease.

This single polypeptide chain forms an active vWF cleaving complextogether with a metal ion selected from the group consisting: Ca⁺⁺,Sr⁺⁺and Ba⁺⁺. The preferred metal ion is Ca⁺⁺. This active complex isable to cleave vWF in a physiological manner as described above.

A further aspect of the present invention relates to an isolated vWFcleavage. complex comprising vWF, a metal ion selected from the groupconsisting of Ca⁺⁺, Sr⁺⁺ and Ba⁺⁺ and one or more single polypeptidechains having vWF protease activity according to the present invention.

The vWF proteolytic activity (also termed “vWF protease activity”) ofthe peptide chains according to the present invention may be determinedby any method described in the art, such as the method according toFurlan et al. (1996), which is preferred for the present invention.Further preferred test systems are described^(,) in InternationalApplication No. WO 00/50904. The test system described in WO 00/50904 isalso suitable for the present vWF protease entities. Alternatively, acollagen binding assay (as described in EP 816 852) can also be used asa test system. Preferably, this test is used for screening anddiagnostic purposes.

A further object of the invention is achieved by providing a compositioncontaining the vWF protease according to the invention together withclusterin or an analog or derivative thereof. With relation to theactivity of the protein, the term “derivative” or “analog” of clusterinrefers to proteins that show the same proteolytic characteristics as thenative clusterin protein.

Clusterin is a heterodimeric glycoprotein consisting of twonon-identical subunits, with a molecular mass of approximately 80 kDa(Rosenberg and Silkensen, J., Int. J. Biochem. Cell Biol., 1995, vol.27, pp. 633-645; Tschopp J. and French, L. E., Clinical and Exp.Immunol., 1994, 97, pp. 11-14). It is produced in a wide array oftissues and found in most biologic fluids. The physiologic functionsdescribed in the prior art include complement regulation, lipidtransport, sperm maturation, initiation of apoptosis, endocrinesecretion, membrane protection and promotion of cell interactions.

It has been found that the unusually high stability of the vWF proteaseof the present invention in circulating plasma is associated with thepresence of clusterin. We have found that the half-life of vWF-cleavingprotease activity in vivo is between 1 and 4 days, while other proteasesin plasma have half-lives in the range of seconds to hours. The ratio ofclusterin to vWF protease in a composition according to the presentinvention is preferably in a range of 10M:1M to 1M:10M, and morepreferably the ratio of clusterin and vWF is in the equimolar range. Inhuman plasma, the concentration of vWF-cleaving protease is 2-10mg/liter whereas that of clusterin is 50-400 mg/liter plasma (the molarratio of vWF-cleaving protease to clusterin in human plasma is about1:20-1:100).

Isolation of the composition from either human plasma or other sources,e.g. supernatants of cell cultures expressing the polypeptide accordingto the present invention, milk or other body fluids of transgenicanimals expressing the polypeptide according to the present inventioncan be performed by chromatographic means. Preferably, the purificationis performed by a combination of chromatographic steps includingimmunoaffinity chromatography, gel filtration, and ion exchangechromatography. For example, the first purification step can beimmunoaffinity chromatography, the second step can be gel filtration,followed by one or more additional immunoaffinity chromatography steps.A further purification can be performed using ion exchangechromatography, preferably anion exchange chromatography and at leastone affinity chromatography. Further purification steps can be performedusing ion exchange chromatography, gel filtration and further affinitychromatography steps.

As an alternative embodiment, the nucleotide sequence as shown in FIG. 2can be used for construoting expression systems providing appropriateelements for the expression of the DNA which can then be used for theexpression of a polypeptide having vWF protease activity according tothe present invention.

The expression vector may comprise, for example, in the direction oftranscription, a transcriptional regulatory region and a translationalinitiation region functional in a host cell, a DNA sequence encoding forthe polynucleotide expressing a vWF protease activity according to thepresent invention and translational and transcriptional terminationregions functional in said host cell, wherein expression of said nucleicsequence is regulated by said initiation and termination regions. Theexpression vector may also contain elements for the replication of saidnucleotide. Examples of DNA expression vectors are pBPV, pSVL, pRc/CMV,pRc/RSV, myogenic vector systems (WO 93/09236) or vectors derived fromviral systems, for example from vaccinia virus, adenoviruses,adeno-associated virus, herpes viruses, retroviruses or baculo viruses.

The expression vector containing the nucleic acid which encodes thepolypeptide having vWF protease activity according to the presentinvention can be used to transform host cells which then produce saidpolypeptide. The transformed host cells can be grown in a cell culturesystem to produce said polypeptide in vitro. The host cells preferablyexcrete the polypeptide having vWF protease activity into the cellculture medium from which it can be prepared.

The host cells may be cells derived from the body of a mammal, forexample fibroblasts, keratinocytes, hematopoietic cells, hepatocytes ormyoblasts, which are transformed in vitro with an expression vectorsystem carrying a nucleic acid according to the present invention andre-implanted into the mammal. The polypeptide according to the presentinvention encoded by said nucleic acid will be synthesized by thesecells in vivo and they will exhibit a desired biological activity in themammal.

The nucleic acid encoding the polypeptide according to the presentinvention may also be used to generate transgenic animals, which expresssaid polypeptide proteins in vivo. In one embodiment of this specificapplication, the transgenic animals may express the polypeptide havingvWF protease activity in endogenous glands, for example in mammaryglands from which the said proteins are secreted. In the case of themammary glands, said proteins having vWF protease activity are secretedinto the milk of the animals from which said proteins can be prepared.The animals may be mice, cattle, pigs, goats, sheep, rabbits or anyother economically useful animal.

The vWF protease composition of the present invention can be used, forexample, to process recombinantly produced vWF. Recombinant vWF (r-vWF)can be produced in CHO cells e.g. according to FEBS. Letter 375, 259-262(1995). The r-vWF recovered in this manner is available as a mature vWFand has a singlet structure, i.e. it differs from plasma-derived vWF,which always has a characteristic satellite structure when examined on2% SDS agarose gels. In International Patent Application No. WO 96/10584describes that r-vWF is comprised of multimers with high structuralintegrity which is retained even after purification and treatment forthe inactivation of viruses. The intact structure of the r-vWF isdefined by a result of electrophoretic analysis consisting of multimerbands with an absence of satellite bands. To prepare an r-vWFpreparation having a structure more closely corresponding to that ofplasma-derived vWF from r-vWF with singlet structure, r-vWF is treatedwith the vWF protease composition of the present invention.

According to a further aspect of the present invention, the amino acidsequence and nucleotide sequence as shown in FIG. 2 can also be used forthe production of a preparation for the prophylaxis and therapy ofdiseases that show supranormal vWF content or an increased level ofhigh-rnolecular weight vWF in patients. This can result in thrombosesand thromboembolic diseases. For example, thrombotic throbocytic purpura(TTP), Henoch-Schönlein purpura, preeclampsia, neonatal thrombocytopeniaor haemolytic-uremic syndrome. By administering an effective dose of apolypeptide having a vWF protease activity and having an amino acidsequence as shown in FIG. 2, this can lead to reduction of the contentof high molecular weight vWF multimers in the patients, resulting ineffective therapy of these diseases. The invention is described in thefollowing examples, without being limited thereto.

EXAMPLES Example 1: Method for Isolating vWF-Cleaving ProteolyticPeptides 1.1. Preparation of an IgG-eTTP-Coupled Affinity Gel

The IgG-eTTP was isolated by aid of a 20 ml protein A-Sepharose®(diameter 1.6 cm) in TBS, pH 7.4.. Pheresis plasma of a patientsuffering from acquired TTP (“erworbenes” TTP; eTTP), which previouslyhad been assayed for its inhibitor content relative to the vWF-cleavingprotease was applied to the column in a volume of 50 ml. Aftersubsequent rinsing with TBS, pH 7.4, the bound IgGs were step wiseeluted with citrate, 0.1 M, pH 4.0, and glycine, 0.1 M, pH 2.7. Thefractions immediately were brought to a physiological pH by adding Tris,1.5 M, pH 8.8, and dialysed against TBS, pH 7.4. The Affi-Gel® Hz wascoupled according to the producer's instructions with the IgG-eTTP whichhad been washed out of the protein A-Sepharose® with a pH of 4.0. Thecolumn material prepared in this manner first was washed as prescribed,subsequently it was washed 3 times alternatingly with 50 ml of buffer Band 200 ml of buffer A (chapter 1.7). Prior to use, intensive rinsingwith buffer A was carried out in each instance.

1.2 First Step

As the starting material, 100 ml of pooled CPD plasma which had comefrom at least three donors and had been stored at −20° C., was usedafter centrifuging at 2,500 rpm (1,100 g) for 5 min. At a relatively lowflow rate (FR: 30 ml/h), the plasma was loaded on a 200 mlchromatographic column with IgG-eTTP Affi-Gel® Hz (hydrazide, diameter2.6 cm) which had been equilibrated in buffer A. After washing with atleast 400 ml of buffer A over night at the same flow rate, a 200 mldesalting gel filtration column (Bio-Gel® P-6DG, diameter 2.6 cm) and a10 ml protein G-Sepharose® (diameter 1.6 cm), which previously also hadbeen rinsed with buffer A, was connected thereto. After the flow ratehad been increased to 100 ml/h, the proteins bound to Affi-Gel Hz wereeluted directly with 50 ml of buffer B onto the Bio-Gel® P-6DG so as toremove from the proteins the NaSCN that had been in buffer B. Theproteins which had been eluted from the desalting column prior to theNaSCN were led through the protein G Sepharose® without interruption,where they were freed from the IgGs. Here, the flow rate was lowered to50 ml/h so as to extend the dwelt time of the proteins in the 10 mlcolumn. For regeneration, the protein G-Sepharose® was shortly washedwith buffer C, and the eluted IgG fraction was stored for analysis.

The first step was carried out 8 times before the collected fractionswhich had been frozen at −20° C. were pooled and further processed.

1.3 Second Step

The pooled fractions from 8 chromatographies of the first step werediluted 1:1 with H₂O so as to obtain an ionic strength at which thedesired proteins would bind to the anion exchange column (High QSupport®). The sample whose volume was from 1,500 to 1,800 ml, dependingon the charge used, was checked for its pH and its ionic strength andapplied over night at a FR of 90 ml/h through a 50 ml column withTherasorb® (diameter 1.6 cm) onto a 5 ml High Q Support® (diameter 1.6cm). Both, Therasorb and High Q Support® had previously beenequilibrated in buffer D. After washing with approximately 150 ml ofbuffer D, the Therasorb was disconnected, and the 25 ml Lentil LectinSepharose® (diameter 1.6 cm) which had been equilibrated in buffer E wasconnected to follow the High Q Support®. At a FR of 60 ml/h, theproteins bound to High Q Support were immediately eluted with buffer Edirectly to the Lentil Lectin-Sepharose®. The proteins which bound tothe Lentil Lectin-Sepharose® could be eluted in two steps with buffers Gand H and could be collected. The proteins which had remained bound toTherasorb and High Q Support® were washed out with buffer C or buffer F,respectively, and discarded after an analysis.

For regeneration, before being used, the Lentil Lectin-Sepharose® ineach case was rinsed according to the producer's instruction 3 timesalternatingly with 20 Mt each of buffers I and J, the High Q Support wasrinsed successively with 10 ml each of NaOH 1N and NaCl 1M.

1.4 Third Step

The pooled fractions which had been eluted from the LentilLectin-Sepharose® with buffer H were dialysed three times for a total:of 4 h, each against 1 l of buffer D, and again applied to the High QSupport at a flow rate of 60 ml/h. Connected thereinfront was a 5 mlheparin-Sepharose (diameter 1.4 cm), which likewise had beenequilibrated in buffer D. After the application of the sample, it wasrinsed with approximately 50 ml of buffer D, the heparin-Sepharose wasdisconnected, and a 500 ml Sephacryl® S-300 HR (diameter 2.6 cm), whichhad been equilibrated in buffer L, was connected thereto. The proteinsbound to High Q Support were directly eluted to the gel filtrationcolumn with 10 ml of buffer K. The exclusion chromatography was effectedat a flow rate of 42 ml/h, and the fractions were collected at 7 mleach. The proteins which were more strongly bound to High Q Support®were again eluted with buffer F, those which remained adhered to theheparin-Sepharose, with buffer K.

1.5 Fourth Step

The pool of the active fractions from the third step was applied withouttreatment at a FR of 10 ml/h to a 1 ml anti-α₂-macroglobulin column(flow rate 0.7 cm) which had been equilibrated in buffer L. Theanti-α₂-macroglobulin column was prepared by immobilization according tothe instructions, of rabbit-anti-α₂-macroglobulin antibodies at aconcentration of 4.9 mg/ml on CNBr-activated Sepharose. The proteinsbound thereon were eluted with NaSCN 3M in buffer L and with buffer Cand stored for analysis.

1.5. Materials

buffer A Tris 10 mM pH 7.4 NaCl 0.15M  Na₃-citrate  1 mM Na acid 0.02%buffer B NaSCN 3.0M pH 7.4 in buffer A buffer C glycine 0.1M pH 2.7 Naacid 0.02% buffer D Tris 10 mM pH 7.4 NaCl 75 mM buffer E Tris 20 mM pH7.4 NaCl 0.5M MnCl₂  1 mM buffer F Tris 10 mM pH 7.4 NaCl 1.0M buffer GTris 20 mM pH 7.4 NaCl 0.5M Methyl-α-D-mannopyranoside 30 mM buffer HTris 20 mM pH 7.4 NaCl 0.5M Methyl-α-D-mannopyranoside 0.3M buffer ITris 20 mM pH 8.5 NaCl 0.5M buffer J Na acetate 20 mM pH 5.5 NaCl 0.5Mbuffer K Tris 10 mM pH 7.4 NaCl 0.5M buffer L (TBS) Tris 10 mM pH 7.4NaCl 0.15M 

Chromatographic Materials

affi-gel hydrazide gel ®: for immobilizing specific Bio-Rad, Hercules,CA, USA IgG's anti-α₂-macroglobulin column: isolation of applicant's ownproduction (see 1.5): α₂-macroglobulinrabbit-anti-human-α₂-macroglobulin antibody on CNBr activated Sepharose4B; 4.9 mg/ml Bio-Gel ® P6-DG, medium: gel filtration Bio-Rad withexclusion limit ≧ 6 kDa CNBr activated Sepharose 4B ®: for AmershamPharmacia Biotech, Uppsala, immobilizing proteins S heparin Sepharose,HITrap ® 5 ml: Amersham Parmacia affinity chromatography: binds variousproteins High Q Support ®, Macro-Prep: strong Bio-Rad anion exchangerIgG-eTTP Affi-gel Hz: for binding vWF-protease applicant's ownproduction (see 1.1.): IgG- eTTP on Affi-gel Hz hydrazide LentilLectin-Sepharose 4B: affinit Amersham Pharmacia chromatography: binds tosugar residues o proteins protein A Sepharose ® CL-4B. binds IgG ofAmersham Pharmacia type 1, 2 and 4 protein G Sepharose ® 4FF: isolationof IgGs Amersham Pharmacia of all types Sephacryl ® S-300 HR: gelfiltration for MWs Amersham Pharmacia 10,000 to 1,500,000 Therasorb:coupled with sheep-anti-human-Ig Serag-Wiessner, Naila, D antibodies:isolation of human immunoglobulins

1.6 Fifth Step

Alternatively, or in addition to step four, an anti-clusterin columnchromatography as a further step can be applied. The samples wereprepared identically to the anti-α2-macrogluobulin-column usinganti-clusterin antibodies.

2. SDS-Page Reduced/Non-Reduced SDS-Polyacrylamide Gel-Electrophoresis(SDS-PAGE)

SDS-PAGE was done according to Lämmli. The separating gels were preparedhaving a size of 13.5 cm height, 15 cm width and 3 mm thickness, havingthe composition as follows:

Concentration of SDS-Polyacrylamide gel for a gradient gel of 4% to 12%:

Acrylamide    4%   12% N,N′-Methylenebisacrylamide 0.107% 0.32% Tris(Tris(hydroxymethyl)aminomethane) 0.4M 0.4M APS(Ammoniumperoxydisulfate)  0.03% 0.03% SDS (Sodium Dodecyl Sulfate) 0.1%  0.1% Temed 0.067% 0.067%  (N,N,N′,N′-Tetramethylethylenediamine)pH 8.7 pH 8.7

30 ml of each of the 4% and 12% solutions, were poured between two glassplates.

After polymerization, the stacking get was prepared, having a height of3 cm and having 16 slots. The volume of each slot was 150 μl.

Stacking Gel

Acrylamide   3% N,N′-Methylenebisacrylamide 0.08% Tris 0.1M APS 0.03%SDS  0.1% Temed  0.2% pH 6.8

100 μl probe were mixed with 50 μl buffer for the SDS-PAG and incubated20 minutes at 60° C. Probes that had to be reduced were mixed with DTT(1,4-Dithio-DL-threitol), 65 mM before the solution was heated up.

Buffer for the SDS-PAGE

Tris 0.15M SDS  4% Glycerin-87% 30% Bromine phenol blue few amounts pH6.8

After heating, the probes were centrifuged, loaded onto the slots of theSDS PAG and covered with electrophoresis buffer. Electrophoresis was runover night in a vertical chamber system, using 60 V voltage.

Electrophoresis Buffer

Tris 50 mM Glycine 0.38M EDTA (Titriplex III: ethylen-  2 mM dinitrilotetra acetic acid- disodium salt -dihydrate) SDS 0.1% standard-pH 8.3

Silver Staining

The gels (13.5×15×0.3 cm) were put into a tray containing a mixture ofmethanol (25%), acetic acid (7.5%), water (65.5%) and glycerin-87% (2%)(fixing solution) for at least 3 hours. Subsequently, the gets wererinsed 4-times with 200 ml water for 1 hour. Then the get was put into asodium thio sulfate solution (0.02%), washed twice with water (1 min)and incubated 2-times 20 minutes with silver nitrate (0.1%) in waterunder constant shaking.

After rinsing with water for two times, the gel was shaken in adeveloping solution, consisting of sodium carbonate (2.5%) andformaldehyde-37% (0.04%) in water. The reaction was stopped with thefixing solution, as soon as the proteins were visible as gray to brownbands.

Example 2: Affinity Purification of von Willebrand Factor (vWF)

The peptide with the sequence AAGGILHLELLV was synthesized on asolid-phase support following the method of Barany, G and Merrifield, R.B. (1980) Solid-phase Peptide Synthesis, in The Peptides vol. 2 (Gross,E. and Meienhofer, J., eds) Academic, New York. After cleavage andde-protection of the peptide, the peptide was purified by ion-exchangechromatography. The peptide was characterized by reverse phase HPLC on aC8 silica column with gradient elution in trifluoro acetic acid withacetonitrile. The peptide showed no major byproducts.

The peptide was solubilized in a concentration of 5 mg/mL in 0.1 molarphosphate buffer pH 7.5 and incubated with a pre-activated gel suitablefor affinity chromatography (Actigel, ALD-Superflow, Sterogene). Priorto coupling of the peptide to the gel the pre-activated matrix wasexcessively washed with the same phosphate buffer. One volume of thepre-washed gel was then mixed with one volume of the peptide solution tobe immobilized and subsequently 0.1 volume portions of a solution of 0.1molar cyanoborohydride (NaCNBH₃) in 0.1 molar phosphate buffer pH 7.5.The gel was suspended in this solution and shaked for 15 hours at roomtemperature. Subsequently the gel was washed on a sinter funnel with a10-fold volume of the phosphate buffer containing 150 mmolar NaCl andwith 5 volumes of the phosphate buffer containing 2 molar NaCl. Then thegel was equilibrated with an access of 0.1 molar phosphate buffer pH7.0.

The gel was then transferred into a chromatographic column having adimension of diameter to get bed height of 1:4. By determining thepeptide concentration the solution of the incubation supernatant afterseparation from the gel and the washing solutions the amount of peptidecoupled to the affinity matrix was calculated. The coupling rate was85%.

The gel was subsequently used to purify vWF from a Factor VIII(FVIII)/vWF complex. A FVIII/vWF complex concentrate was producedaccording to EP 0 270 516 containing vWF in a concentration of 260 UvWF:Ag/ml and a specific activity of 13.5 U vWF:Ag/mg Protein. Theconcentrate was diluted with 20 mM phosphate buffer pH 7.0 to a finalvWF concentration of 6 U vWF:Ag/mL. A volume of 20 ml of this solutionwas subjected to the affinity column with immobilized peptide describedabove. After washing the column with 10 ml of the phosphate buffer thevWF specifically bound to the peptide ligand was eluted by a lineargradient from 0-2 mol/l NaCl in phosphate buffer at a flow rate of 1ml/minute. Fractions of 1 ml were collected and their optical densitywas determined at 280 nm. All fractions were measured for their contentof vWF antigen determined by a specific ELISA method (Asserachrom vWF,Boehringer, Mannheim). Measurement showed a specific peak of vWF elutingfrom the peptide at a NaCl concentration of 100 mmol/l, while most ofthe protein measured by UV-absorption eluted prior to the vWF fractionwith the washing buffer. The vWF containing fractions were pooled andmeasured for vWF activity. The vWF in this pool had a specific activityof 95 U vWF/mg protein and was essentially free from other proteins.

Example 3: Anti-Peptide Antibodies

The peptide with the sequence AAGGILHLELLV was synthesized and purifiedas described in example 2. The peptide was then used to immunize 3months old BALB/c mice with the following protocol: A primarysubcutaneous injection of 100 μg peptide antigen emulsified in Freund'scomplete adjuvant in 100 μl followed by intra-peritoneal boosts of 100μg peptide antigen in phosphate buffered saline at monthly intervals.

The anti-peptide titer was tested by routine ELISA method using.purified peptide as screening antigen. After the final boost the spleenswere taken from the mice for dell fusion. Cell fusion was carried outaccording to a standard protocol originally described by Kohler G. andMilstein C. 1975, Nature 256:495. Anti-peptide antibodies producinghybridoma cell lines were screened by standard techniques with thepurified peptide as screening antigen essentially based on aconventional ELISA methodology. After cloning a cell line could beisolated with a high expression level of an antibody specific for thescreening peptide with the sequence AAGGILHLELLV. This cell line wascultured on serum-free culture medium and grown to high density. Thesupernatant of the cell culture was harvested by centrifugation toremove cells and the monoclonal antibody containing supernatant wasconcentrated by ultra-diafiltration and conditioned for further use.

The monoclonal antibody obtained had a high selectivity for the vWFcleaving protease as described by Furlan et al. 1996, Blood87:4223-4234. This monoclonal antibody was immobilized to a polystyreneELISA plate in a carbonate/bi-carbonate buffer, 0.05 molar, pH 9.6, at aconcentration of 5 μg immunoglobuline/ml overnight (16 hours) at 4° C.,with each 100 μl of coating solution per well. The coating solution wasremoved from the wells and replaced by a solution of bovine serumalbumin (BSA) at a concentration of 100 μg/ml at a volume of 100 μL perwell, for 2 hours. The BSA solution was removed and the wells werewashed with phosphate buffered saline. The pre-coated plates were thenincubated with either samples of platelet poor plasma from healthy humanplasma donors or platelet poor plasma from patients with an uncleardiagnosis of either thrombotic thrombocytopenic purpura (TTP) orhemolytic uremic syndrome (HUS). After incubation of the plasma sampleswith the antibody coated ELISA plates as in a routine sandwich ELISAsystem, after 3 hours the plasma was removed from the wells. Wells werewashed with phosphate buffered saline and incubated with the monoclonalantibody directed against the peptide with the sequence AAGGILHLELLV,conjugated with horse radish peroxidase following the method of Wilson,M. B. and Nakane, P. K. (1978) In Immunofluorescence and RelatedStaining Techniques. Knapp, W. Holubar, K. and Wick, G (eds),Elsevier/North Holland, Amsterdam, p. 215, and detected by the OPTreagent as described by Cathy D. and Raykundalia Ch. (1989) ELISA andrelated enzyme immunoassays, in Antibodies II a practical approach.Cathy D (ed), IRL Press Eynsharri Oxford England, p. 97.

Based on the level of the samples from the healthy human plasma donors anormal range was established. Plasmas from patients with HUS had a vWFprotease activity equivalent to healthy humans while patients with TTPhad a decreased protease activity as confirmed by an assay based on adifferent assay principle as described in WO00/509904.

Example 4: Amino Acid Sequencing and Amino Acid Analysis of the vWFProtease

The final protein preparation from the third step of isolation waselectrophoresed on a 1.5 mm-thick SDS-polyacrylamide gel according toLaemmli (Laemmli UK., Nature, 1970, 227, pp. 680-685) A gradient of 4 to12% polyacrylamide was used for fractionation of high molecular weightproteins, and a gradient of 8 to 12% polyacrylamide for low molecularweight proteins. After electrophoresis under non-reducing or reducingconditions (final concentration 65 mmol/l dithiotreitol), the proteinswere blotted onto PVDF-membranes and stained, for 2 min with 0.25%Coomassie Blue in 45% methanol, 9% acetic acid and 46% H₂O. Afterrinsing with: a Mixture of 50% methanol, 10% acetic acid and 40% H₂O,the visible protein bands were cut out and analyzed on a Procise-cLCSequencer (Foster City, Calif.) at the Chemical Institute of theUniversity of Bern.

The N-terminal amino acid sequence of polypeptide bands separated bySDS-PAGE of purified vWF-cleaving protease is shown in table 1:

Molecular weight Amino Acid sequence 350 kDa unreducedSer-Val-Ser-Gly-Lys- Pro-Gln-Tyr-Met-Val* 150 kDa unred.Ala-Ala-Gly-Gly-Ile 140 kDa unred. Ala-Ala-Gly-Gly-Ile 130 kDa unred.Ala-Ala-Gly-Gly-Ile 110 kDa unred. Ala-Ala-Gly-Gly-Ile- Leu-His-Leu-Glu 70 kDa unred. Asp/Ser-Gln/Leu-Thr/ Met-Val/Pro-Ser/Phe**180 kDa reduced Ala-Ala-Gly-Gly-Ile- Leu-His-Leu-Glu 170 kDa reducedAla-Ala-Gly-Gly-Ile 160 kDa reduced Ala-Ala-Gly-Gly-Ile-Leu-His-Leu-Glu-Leu- Leu-Val-Ala-Val-Gly 120 kDa reducedAla-Ala-Gly-Gly-Ile- Leu-His-Leu-Glu-Leu- Leu-Val-Ala-Val-Gly 40 kDa reduced Asp-Gln-Thr-Val-Ser** *identified as α2-macroglobulin**identified as clusterin

Analysis of the composition of the amino acids was performed from thesame sample as used for amino acid sequencing. The protein bands werehydrolyzed in the gas phase over 6 N HCl for 22 hours' at 110° C. andthe amino acids were determined by high-performance liquidchromatography. Four unreduced polypeptide bands from SDS-PAGE ofpurified vWF-cp with M_(r) 150, 140, 130, and 110 kDa were analyzed

The results are shown in Tab. 2:

No. residues/100 residues Amino acid 150 kDa 140 kDa 130 kDa 110 kDa Asx6.7 7.0 7.4 8.3 Glx 12.2 12.0 12.8 11.8 Ser 8.4 8.9 8.8 9.2 Gly 11.812.1 12.1 12.9 His 2.5 2.3 2.5 2.4 Arg 8.3 7.6 8.1 7.2 Thr 5.6 5.5 5.65.7 Ala 10.1 9.5 9.6 8.2 Pro 8.9 8.5 8.3 8.1 Tyr 2.1 2.7 2.3 2.4 Val 7.06.9 6.7 6.5 Ile 2.7 2.9 2.6 3.0 Leu 10.0 9.9 9.1 9.7 Phe 2.6 2.8 2.6 3.2Lys 1.0 1.3 1.3 1.4

Example 5: Search on Chromosome 9 Clone RP11-224N20 for Potential Exonsof the vWF-Protease

The coding region of the N-terminus of the activated vWF protease(aminoacids A-A-G-G-I-L-H-L-E-L-L-V-A-V-G) was found on Chromosome 9clone RP11-224N20 bases 156653 to 156697. Thus the nucleotide sequencefrom base 150001 to 185911 was screened for potential exons. Consecutiveoverlapping genome-segments with various lengths (1500 bases-5000 bases)were analysed using search engines that were queried via theinternet-explorer. The genomic sequence segments, its translations andthe results of the search were managed using the ‘Vectors NTI Suite1v.5.2’ computer-program (Informax Inc., USA).

The first four exons of the search were translated into thecorresponding amino add sequence and these sequences were searched forhomologies. This search revealed that each sequence displays highhomology with the amino acid sequence of the family of human disintegrinand metalloproteinases with thrombospondin motifs (ADAM-TS).

By alignments performed with the ‘Vectors NTI Suite1 v.52-AlignX’program (Informax Inc., USA), further potential exons were identifiedthat encode for amino acid sequences that are homologous to segments ofthe human ADAM-TS sequences. Exons potentially encoding for the entireproteinase domain, for parts of the disintegrin like domain, thethrombospondin motif, the cystein-rich domain, as well as for threepotential thrombospondin submotifs or a new ADAM-TS proteinase were thusidentified. The succession of the exons on the genomic sequence wasfound to be consistant with the succession of the corresponding aminoacid segments on the ADAM-TS consensus sequence.

Example 6: Cloning of the Gene Expressing the vWF-Protease Gene

Human salivary gland poly A+ RNA was purchased from Clontech. Firststrand cDNA was, obtained using Expand reverse transcriptase (Roche) andoligo d(T) primer according to the manufacturer's instructions. PCR wasperformed using 5′CGGCGGGATCCTACACCTGG 3′ and 5′AATGGTGACTCCCAGGTCGA 3′as primers with 10 ng of salivary gland cDNA as template and 10 U of HotStar Tag polymerase (Quiagen). The thermal cycling parameters were aninitial incubation at 94° C. for 15 minutes followed by 45 cycles of 94°C. (50 sec), 50° C. (50 sec), 72° C. (2 min). PCR products were directlysequenced in both directions using the BigDye Terminator CycleSequencing Ready Reaction Kit (PerkinElmer Life Science).

The obtained DNA sequence was used to scan the genomic data base usingBLAST (basic local alignment search tool) programs and matched to thechromosome 9 clone RP11-224N20. DNA sequence was translated to aminoacids sequence using ExPASy proteomic tools. The DNA and translatedamino acid sequences corresponding to 4 putative exons of chromosome9q34 are shown in FIG. 2.

What is claimed:
 1. An pharmaceutical composition comprising aneffective dose of a recombinant polypeptide for the treatment of adisease in which the patient has a supranormal vWF content or anincreased level of high-molecular weight vWF, wherein the polypeptideexhibits vWF protease activity and vWF binding activity, is produced inan in vitro mammalian cell culture system, and has a molecular weightbetween about 180 kD and about 120 kD as determined by SDS-PAGE underreducing conditions and comprising the amino acid sequence(SEQ ID NO: 4) AAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCL ITEDTGFDLGVTI.


2. The pharmaceutical composition of claim 1, wherein the in vitromammalian cell culture system is a CHO cell culture system.
 3. Thepharmaceutical composition of claim 1, wherein the disease is thromboticthrombocytopenic purpura (TTP), Henoch-Schonlein purpura, preeclampsia,neonatal thrombocytopenia, or hemolytic-uremic syndrome.
 4. Thepharmaceutical composition of claim 1, wherein the recombinantpolypeptide comprises the amino acid sequence: (SEQ ID NO: 4)AAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCL ITEDTGFDLGVTI.


5. The pharmaceutical composition of claim 3, wherein the disease isthrombotic thrombocytopenic purpura (TTP), Henoch-Schonlein purpura,preeclampsia, neonatal thrombocytopenia, or hemolytic-uremic syndrome.6. A method of treating a disease in which a patient has a supranormalvWF content or an increased level of high-molecular weight vWF, whereinthe method comprises administering a pharmaceutical compositioncomprising an effective dose of an isolated polypeptide that has vWFprotease activity and comprises a peptide chain that has a molecularweight from about 180 kD to about 120 kD as determined by SDS-PAGE underreducing conditions and comprises the amino acid sequence (SEQ ID NO: 4)AAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCL ITEDTGFDLGVTI.


7. The method of claim 7, wherein the disease is thrombotic throbocyticpurpura (TTP), Henoch-Schonlein purpura, preeclampsia, neonatalthrombocytopenia or hemolyticuremic syndrome.